Adult T-cell leukemia (ATL) is a very aggressive and often fatal T-cell malignancy that is caused by infection of the type I human T-cell leukemia virus (HTLV-I). The HTLV-I encoded Tax protein appears to play a central role in the initiation of this virally induced T-cell malignancy. Tax acts by activating cellular transcription factors, including members of the NF- 1kappaBalpha/Rel family, which in turn induces various cellular genes involved in lymphocyte activation and growth. In resting T cells, NF- kappaB/Rel factors are sequestered m the cytoplasm by various inhibitory proteins including I-kappaB-alpha, a 37 kD protein containing multiple ankyrin-like repeats. HTLV-I Tax expression results in the constitutive nuclear expression of these kappaB-enhancer binding proteins, leading to the deregulated expression the various cellular growth-related genes, which have been proposed to induce the polyclonal T-cell proliferation, a prelude to the establishment of ATL. Our recent studies suggest that Tax activation of NF-kappaB is associated with the phosphorylation and degradation of I-kappaB-alpha and the activated nuclear NF-kappaB seems, together with other yet to be identified transcriptional enhancers or silencers, to mediate the Tax-induced transcriptional induction of the c- rel proto-oncogene. We have further observed that these Tax-induced cellular events can be blocked by various inhibitors of cellular signal transduction. Together, these results raise the possibility that Tax may induce a specific cellular signal transduction pathway leading to a cascade of both cytoplasmic and nuclear reactions with phosphorylation of IkBa serving as a molecular trigger. This specific action of Tax results in the nuclear expression of both NF-kB and c-Rel. Based on these results, the overall objective of this grant proposal is a fundamental understanding of the Tax-mediated NF-kappaB/Rel induction signaling pathway. To approach this overall objective, a set of biochemical studies will be undertaken to precisely map the sites of Tax-induced phosphorylation within I-kappaB-alpha. In turn, site-directed mutations will be sequentially introduced into these sites to examine the functional significance of these phosphorylation events on Tax-mediated I-kappaB- alpha degradation. In conjunction with these site-directed mutagenesis, progressive deletional analyses will also be performed to completely define the sequences within I-kappaB-alpha that are required for protease attack. To explore the Tax-mediated signaling pathway, different strategies will be used to identify and characterize the cellular molecular components, including I-kappaB-alpha-specific kinases and the more proximal signaling molecules, involved in the Tax-mediated activation of NF-kappaB/Rel. Finally, the precise mechanism of Tax-induced transcriptional induction of c-rel gene will be explored by isolation and functional analyses of the human c-rel gene promoter.